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. 2017 Nov;16(5):7432-7438.
doi: 10.3892/mmr.2017.7546. Epub 2017 Sep 20.

Vitamin D Alleviates Lipopolysaccharide‑induced Acute Lung Injury via Regulation of the Renin‑angiotensin System

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Free PMC article

Vitamin D Alleviates Lipopolysaccharide‑induced Acute Lung Injury via Regulation of the Renin‑angiotensin System

Jun Xu et al. Mol Med Rep. .
Free PMC article

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. ALI and ARDS result are associated with high mortality in patients. At present, no effective treatments for ALI and ARDS exist. It is established that vitamin D exhibits anti‑inflammatory effects, however, the specific effect of vitamin D on ALI remains largely unknown. The aim of the present study was to investigate whether, and by which mechanism, vitamin D alleviates lipopolysaccharide (LPS)‑induced ALI. The results demonstrated that a vitamin D agonist, calcitriol, exhibited a beneficial effect on LPS‑induced ALI in rats; calcitriol pretreatment significantly improved LPS‑induced lung permeability, as determined using Evans blue dye. Results from reverse transcription‑quantitative polymerase chain reaction, western blotting and ELISA analysis demonstrated that calcitriol also modulated the expression of members of the renin‑angiotensin system (RAS), including angiotensin (Ang) I‑converting enzymes (ACE and ACE2), renin and Ang II, which indicates that calcitriol may exert protective effects on LPS‑induced lung injury, at least partially, by regulating the balance between the expression of members of the RAS. The results of the present study may provide novel targets for the future treatment of ALI.

Figures

Figure 1.
Figure 1.
Cal inhibits ACE and AT1R expression, and induces ACE2 expression in LPS-treated rat PMVECs. (A) Morphology of rat PMVECs and immunostaining for PMVEC marker FITC-PHA. Phase: Normal PMVEC morphometrics under phase-contrast microscopy (magnification, ×200). The cells grew initially as capillary-like structures and assumed typical cobblestone morphology of endothelial cells at confluence. PHA: PMVECs bound to FITC-PHA under fluorescence microscopy to reveal yellow green fluorescence (magnification, ×400). Reverse transcription-quantitative polymerase chain reaction analysis of (B) ACE and ACE2, and (C) AT1R and AT2R mRNA expression levels in rat PMVECs in various treatment groups. ACE2 is a counter-regulator of ACE and AT1R is a downstream effector of ACE. AT2R was employed as a control. (D) Western blot analysis of the protein levels of ACE, ACE2, AT1R and AT2R in rat PMVECs in various treatment groups. (E) Densitometric analysis of the relative protein expression levels of ACE, ACE2, AT1R and AT2R. Data are presented as the mean + standard deviation of three biological replicates. *P<0.05 and **P<0.01 vs. NT; #P<0.05 and ##P<0.01 vs. LPS-only. Cal, calcitriol; Ang, angiotensin; ACE, Ang I-converting enzyme; AT1R, Ang II type 1 receptor; LPS, lipopolysaccharide; PMVECs, pulmonary microvascular endothelial cells; AT2R, Ang II type 2 receptor; NT, no treatment; FITC-PHA, fluorescein isothiocyanate-endothelial marker protein phy agglutinin.
Figure 2.
Figure 2.
Cal suppresses REN and Ang II levels in LPS-treated rat PMVECs. (A) RT-qPCR analysis of REN and Ang II mRNA expression in rat PMVECs in various treatment groups. (B) Levels of REN and Ang II in the culture medium of rat PMVECs were quantified by ELISA. Data are presented as the mean + standard deviation of three biological replicates. *P<0.05 and **P<0.01 vs. NT; #P<0.05 and ##P<0.01 vs. LPS-only. Cal, calcitriol; REN, renin; Ang, angiotensin; LPS, lipopolysaccharide; PMVECs, pulmonary microvascular endothelial cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NT, no treatment.
Figure 3.
Figure 3.
Cal reduces ACE and AT1R expression, and increases ACE2 expression in LPS-treated rat lung tissue. (A) Number of cells in rat bronchoalveolar lavage fluid collected from rat lung tissues in various treatment groups. (B) Results of Evans blue permeability assays in rat lung tissues from various treatment groups. Reverse transcription-quantitative polymerase chain reaction analysis of (C) ACE and ACE2, and (D) AT1R and AT2R expression in rat lung tissues from various treatment groups. (E) Western blot analysis of the protein levels of ACE, ACE2, AT1R and AT2R in rat lung tissues from various treatment groups. (F) Densitometric analysis of the relative protein expression levels of ACE, ACE2, AT1R and AT2R. Data are presented as the mean + standard deviation of three biological replicates. *P<0.05 and **P<0.01 vs. NT; #P<0.05 and ##P<0.01 vs. LPS-only. Cal, calcitriol; Ang, angiotensin; ACE, Ang I-converting enzyme; AT1R, Ang II type 1 receptor; LPS, lipopolysaccharide; AT2R, Ang II type 2 receptor; NT, no treatment.
Figure 4.
Figure 4.
Cal impairs the induction effects of LPS on REN and Ang II levels in rat lung tissue. (A) RT-qPCR analysis of REN and Ang II mRNA expression in rat lung tissues from various treatment groups. (B) Levels of REN and Ang II in bronchoalveolar lavage fluid from various treatment groups were quantified by ELISA. Data are presented as the mean + standard deviation of three biological replicates. *P<0.05 and **P<0.01 vs. NT; #P<0.05 and ##P<0.01 vs. LPS-only. Cal, calcitriol; LPS, lipopolysaccharide; REN, renin; Ang, angiotensin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NT, no treatment.

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